We are seeking principles which govern biological activation of molecular oxygen. Our objective is a generalized understanding of mechanisms by which oxygen supports life in terms of structures of oxygenases and oxidases, oxygen, cofactors, and substrates, and the mechanisms of oxygen-activating reactions. We propose to investigate selected dioxygenase, mixed-function oxidases, and oxidase reactions in which copper, iron (non-heme) and heme prosthetic groups. We will continue our biochemical and biophysical studies by (1) determining structures of active sites and related protein structures of copper, heme, and iron oxygenases and mixed-function oxidases, (2) characterizing reactions which take place between these oxygenases, their substrates, and O2, in terms of oxygen- and substrate-containing intermediates, (3) detecting and characterizing intermediates by cryoenzymological techniques, (4) testing enzymic relevance of reaction intermediates by specific transfer of (isotopic) oxygen to substrates, (5) establishing by kinetic and thermodynamic studies the sequence of formation and reaction of intermediates, and (6) elaborating principles which govern biological activation of oxygen.